LabMed

Sickle Cell Trait

At a Glance

Sickle cell trait may be expected in any person with a family history of sickle cell trait or sickle cell disease. This mutation is commonly found in persons of African, Middle Eastern, or Mediterranean descent, but rare cases have been reported in most ethnic groups. It is found in approximately 8% of Americans of African descent.

Hemoglobin S arises from a mutation in the hemoglobin β-globin gene, which decreases hemoglobin solubility in its deoxygenated state. Deoxygenated mutant hemoglobin, if present in sufficient amounts, forms polymers that contort the red cell into the characteristic sickled shape. Sickle disease (two mutated genes) carries significant morbidity and mortality from this phenomenon.

In contrast, individuals with sickle cell trait (a single mutated gene) are usually asymptomatic, with normal hematologic indices and blood smear. Here, only about 40% of hemoglobin is in the mutant form; there is enough normal hemoglobin to prevent sickling.

Significant hemolysis and/or anemia or microcytosis with sickle cell trait should prompt further investigation for the coinheritance of a thalassemia or other hemoglobinoapthy.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

If either the sickling test is positive or a variant hemoglobin is suspected, iso-electric focusing (IEF) or electrophoresis (EP) of hemoglobin dimers should be ordered.

Individuals with sickle cell trait demonstrate normal RBC indices, a positive sickling tests, and about 40% hemoglobin S by HPLC or CEP. Any difference from this pattern of results should prompt consideration of another hemoglobinopathy, such as sickle cell disease or a concurrent β-thalassemia.

Assessment of iron status, which is important in anemia, is usually accomplished through tests for ferritin and transferrin saturation (<20 ng/mL and<15%, respectively, in uncomplicated iron deficiency). Sickle anemia is an iron-loading condition, particularly if transfusions are required. Ferritin should be monitored and the value kept below 1000 ng/mL by the use of chelators like Exjade (Table 1).

Table 1

Sickling Test

Hemoglobin HPLC or CE

Positive

~40% hemoglobin S, ~60% hemoglobin A

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Patients with sickle cell trait should have normal red cell morphology. However, extreme conditions, including high-altitude exercise, severe pneumonia, and flying in an unpressurized aircraft, have been associated with sickling complications in persons with sickle cell trait.

In some rare cases (<1%), hematuria may be seen in patients with sickle cell trait.

Iron deficiency can lower the percentage of hemoglobin A2, which may mask a concurrent β-thalassemia. A mean corpuscular volume (MCV)/RBC less than 14 is highly suggestive of β-thalassemia.

If the value of hemoglobin A2 is used as a key indicator of β-thalassemia, it is crucial to exclude the presence of hemoglobin A2′. This delta chain variant is clinically benign but will be present at equal concentration to hemoglobin A2; to obtain an accurate value of delta chain concentrations, hemoglobins A2 and A2′ must be added together. It can be difficult to visualize hemoglobin A2′ on EP or IEF since the percentage is small and it coelutes with hemoglobin S on HPLC.

Glycated hemoglobin S elutes with hemoglobin A2 on HPLC and may falsely elevate the value of hemoglobin A2, leading to erroneous suspicion of β-thalassemia.

Transfusion is always assumed with 95% hemoglobin A, although occasionally hemoglobin C- or D-trait blood is transfused, which results in unexpected hemoglobin variants. Although sickle cell trait is not a transfusion-dependent state, a patient may receive transfusions for other reasons, such as traumatic blood loss.

What Lab Results Are Absolutely Confirmatory?

The demonstration of a substitution of valine for glutamic acid at position 6 of the β-globin chain is diagnostic for hemoglobin S(β6Glu→Val). This can be confirmed by genetic testing, although the expense is rarely justified. The sickle mutation is at the same location as hemoglobin C, so when both hemoglobin S and C are present, these mutations must be one on each β-globin gene.

In practice, however, the demonstration of a positive sickling test, HPLC peaks in the A- and S-windows, and bands in the hemoglobin A and S positions on EP or IEF is considered confirmatory for sickle cell trait. The percentage of hemoglobins F and A2 will be within normal limits.

In sickle cell trait, hemoglobin S is always lower than that of hemoglobin A (typically 35-40%) because there is reduced affinity of the mutant β-globin chains for normal α-globin chains. Deviation from the 35-40% of hemoglobin S or the presence of an unusually large amount of hemoglobin F is suggestive of a different hemoglobinopathy, a concurrent α- or β-thalassemia, and/or iron deficiency.

Many newborn screening programs include tests for common hemoglobinopathies and successfully identify sickle cell trait. The expected pattern is F-A-S. The relative percentage of hemoglobins S to A should approximate that seen in adults.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

If the severity of the clinical presentation does not match the initial diagnosis, sequencing of the α- and/or β-globin genes (including upstream regulatory sequences) may be necessary in order to arrive at a definitive diagnosis. The presence of hemoglobin H may indicate a 3-gene α-thalassemia (or a 2-gene α-thalassemia in a neonate). An elevated percentage of hemoglobin A2 is indicative of a β-thalassemia in a nondiabetic patient.

An elevated percentage of hemoglobin F is suggestive of an α-thalassemia, β-thalassemia, hemoglobin S/C disease, or hereditary persistence of fetal hemoglobin. Treatment with hydroxyrea increases the percentage of hemoglobin F and has an ameliorating effect on sickling. It is also used for polycythemias and as a chemotherapeutic agent.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Hemoglobins D-Los Angeles and G-Philadelphia migrate with hemoglobin S on some EP methods, but not HPLC; neither gives a positive sickling test.

The current generation of hemoglobin A1C (glycated hemoglobin) assay has eliminated previously observed unreliability in the presence of hemoglobin S-trait, so A1C can be interpreted with confidence in these patients.

The sickling test is a screening tool that detects any hemoglobin that polymerizes under reduced oxygen tension. Therefore, this assay cannot differentiate among sickle cell trait, sickle cell disease, and a compound heterozygote (e.g., hemoglobin SC). All results should be confirmed by additional testing, especially if they do not agree with the clinical picture. Where patients’ percentages of hemoglobins S, A, and F differ from those expected in uncomplicated presentations, considerations of alternative hemoglobin variants is important. Other hemoglobins that also give positive sickling tests may need to be considered. With the exception of C-Harlem, most of these are rare or isolated reports. With the exception of Porto-Alegre, all contain the S mutation (ß6 Glu → Val) in addition to the following mutations:

Hgb C-Harlem (ß73 Asp → Asn) (C-Georgetown)

Hgb C-Ziquinchor (ß58 Pro → Arg)

Hgb S-Oman (ß121 Glu → Lys)

Hgb S-Providence (ß82 Asn → Asp)

Hgb S-Travis (ß121 Ala → Val)

Hgb Jamaica Plain (ß68 Leu → Phe)

Hgb Antilles (ß23 Val → Ile)

Hgb Porto-Alegre contains only the (ß9 Ser → Cys) mutation

The sickling test may give a false negative if the hemoglobin S concentration is less than 1 g/dl (typically <10-15% of the total hemoglobin). This may occur following transfusions or in cases of high hemoglobin F (e.g., neonates and hereditary persistence of fetal hemoglobin).

The sickling test may give a false positive if there are nucleated RBCs in the peripheral blood or if the patient has a marked hypergammaglobulinemia (e.g., multiple myeloma).

There are many causes of RBC hemolysis other than hemoglobinopathies, some include:

Anemia of inflammation (anemia of chronic disease) has a normal to elevated ferritin, and further tests are required to determine whether iron deficiency is also present. Iron overload is indicated when the transferrin saturation is greater than 75%. If iron overload is already established, monitoring liver function may be indicated, and the physician should be attuned to monitoring for other organ damage (Table 2).